High oleic high stearic plants seeds and oils

ABSTRACT

The invention relates to plant seeds that contain an oil having an oleic acid content of more than 40 wt % and a stearic acid content of more than 12 wt % based on the total fatty acid content of said oil, and wherein a maximum of 10 wt % of the fatty acid groups in the  sn -2 position of the TAG molecules constituting the oil are saturated fatty acid groups. The invention also relates to plants that can be grown from the seeds, oil that can be extracted from the seeds, and to methods for obtaining the seeds, plants and oil.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is the National Stage of International Application No.PCT/EP00/05150, filed Jun. 5, 2000, which claims the benefit of U.S.Provisional Application No. 60/180,455, filed Feb. 4, 2000, and which isa continuation-in-part of U.S. application Ser. No. 09/326,501, filedJun. 4, 1999 (now U.S. Pat. No. 6,388,113).

FIELD OF THE INVENTION

The present invention relates to new seeds that contain an oil having ahigh oleic and high stearic content. The invention also relates toplants producing these seeds and to the oil that is contained in theseeds. In addition, the invention relates to methods for producing theseeds, plants and oil.

BACKGROUND OF THE INVENTION

The uses of oils are determined by their fatty acid composition. Theprincipal component of oils are the triacylglycerol (TAG) molecules,which constitute normally more than 95% of the oil. Three fatty acidsare bound to a molecule of glycerol to make the TAG. If these fattyacids are mainly saturated fatty acids (“saturates”) the product iscalled fat and it is solid at room temperature. On the other hand if thefatty acids are mainly unsaturated then it is called oil and it isliquid at room temperature.

The oils obtained from seeds cultivated in temperate climate (sunflower,soybean, rapeseed, etc.) have mainly unsaturated fatty acids, likelinoleic and oleic acids, so they are liquid and primarily used forcooking, salad dressing, etc. Fats are obtained from animals (margarine,lard, etc.), some tropical trees (cocoa, palm) or chemically modified(hydrogenation and transesterification) liquid vegetable oils. They havemainly saturated (palmitic or stearic acids) or chemically modifiedfatty acids (trans fatty acids) all with high melting point.

Table 1 shows as an example the fatty acid composition and otherproperties of some fats and oils. The fats are needed for most of thefood industry to make margarine, shortening, bakery, confectionery,snacks, etc. The food industry uses the fat for these purposes becauseof their plastic properties (they do not melt, can be spread, or do notstick to the hand) and stability (they have a good resistance tooxidation at room or high temperatures).

TABLE 1 Oil or Fatty acid composition (%) Properties fat Others¹Myristic Palmitic Stearic Oleic Linoleic Trans Saturated Lard 3 2 25 1245 10 1 79  Butter 14 10 26 12 28 3 3 84  Margarine 10 7 46 34 23 — Palmoil 1 45 5 39 9 18  Olive oil 1 14 3 71 10 2 Cocoa 26 35 35 3 4 butterNormal 7 5 30 57 1 sunflower High 5 4 88 2 1 oleic sunflower ¹“others”are palmitoleic in the case of lard and olive oil and also fatty acidsshorter than 12 carbons in butter *depends on the level of hydrogenation

The actual available fats are however not a good option because theyhave negative nutritional properties. The main problem is that theyraise the bad form of serum cholesterol (low density lipoprotein, LDL).This is due to several facts, some related to the origin of the fat andothers with the manipulation thereof. Animal fats have most of thesaturated fatty acids in the position 2 of the TAG molecule. Mostvegetable fats and oils, however, have only minor amounts of saturatedfatty acids in this position and are therefore more healthy.

During digestion the TAG molecule is hydrolysed by enzymes calledlipases (FIG. 1). The fatty acids in positions 1 and 3 are liberated asfree fatty acids. If these fatty acids are saturated they form insolublesalts with calcium and magnesium, being mostly excreted. But fatty acidsin position 2 form with the glycerol a molecule of monoacylglycerol,which has detergent properties and is easily absorbed into the body. Thesaturated fatty acids from animal fats are then absorbed, thus raisingLDL.

In order to increase the percentage of saturated fatty acids, vegetableoils are hydrogenated and/or transesterified. The hydrogenation processproduces trans fatty acids that probably are even worse than saturatedfatty acids as illustrated by Willett, W. C. & Ascherio, A. (1994) Transfatty acids: Are the effects only marginal? American Journal of PublicHealth 84:722–724. The transesterification process changes randomly thefatty acids within the three positions, converting a healthy vegetableoil with low saturated fatty acid in the 2 position in an oil that hasnear 30% of saturated fatty acids. So neither of the two chemicalmodifications leads to a healthy product.

However, not all fats are unhealthy. It has been demonstrated that cocoabutter, which has around 60% of saturated fatty acids, the rest beingmainly oleic acid, does not raise serum cholesterol. This is due to twomain reasons. One is that only 4% of the saturated fatty acids are inposition 2 and the other is that the principal saturated fatty acid isstearic acid. Stearic acid does not have a negative effect on serumcholesterol. Probably the amount of 35% of oleic acid in the cocoabutter also adds to its healthy property.

It is important to note that except in cocoa butter, palmitic acid isthe main saturated fatty acid of commodity fats. Palmitic is however nota very healthy fat.

Traditional breeding and mutagenesis has not been the only tool used toform seeds producing oil with different fatty acid profiles. Increasesin stearic acid in oil bearing plants have also been addressed by theintroduction of transgenes into the germplasm, to alter the fatty acidbiosynthesis pathway of the vegetable oil. The fatty acid biosynthesisin vegetable oil, but more particularly sunflower oil, includes thebiosynthesis of basically two saturates (palmitate, stearate) and twounsaturates (oleate and linoleate). In oilseeds, the stearoyl-ACPdesaturase is the enzyme which introduces the first double bond onstearoyl-ACP to form oleoyl-ACP. Thus, this is an enzyme that assists inthe determination of the unsaturation in the C18 length fatty acids.

In U.S. Pat. No. 5,443,974 the inhibition of canola enzyme stearoyl-ACPdesaturase was described. The stearate levels were increased but thelevels of palmitate were basically unaffected. Inhibition of the plantenzyme stearoyl-ACP desaturase in canola was also reported by Knutzon etal., Proc. Natl. Acad. Sci. USA 89:2624–28 (1992). These results showedan increase in the level of stearate produced in the canola seed. Theresearch also showed that inhibition by antisense in seeds of canola andsoybean, respectively, showed increased stearate. When a plasmidcontaining a gene encoding for stearoyl-ACP desaturase was placed incanola, this inhibition resulted in an increase in stearic acid butunfortunately a reduction in the oleate. However, in the soybean thisinhibition of stearate resulted in a less dramatic reduction of theoleate. This slower decrease in oleate however may have been a functionof the small initial levels of oleate in the soybean. The fatty acidpathway in most oilseed plants appears to be resistant to maintainingboth oleic and stearic at elevated levels.

PCT/US97/01419 describes increased levels of both stearic acid andpalmitic acid in sunflowers through the inhibition of the plant enzymestearoyl-ACP desaturase. As indicated above, palmitic oil is not,however, viewed as being a very healthy oil.

PCT/US96/09486 discloses that sunflower oil levels of both palmitic andoleic acids could be increased, the seeds having increased levels ofpalmitic acid of 21–23% and of oleic acid of 61%. The sunflower oil isliquid at room temperature. But the increased palmitic fatty acid levelis alleged to allow the oil to be used in shortening and in margarinewith relatively low level of hydrogenation, which leads to a relativelylow level of trans-fatty acids in the resulting product. However, thecommercial value may be questioned because of the high level of palmiticacid.

There thus remains a need for a sunflower oil which is both healthy anduseful for industrial purposes. Furthermore, it is desirable to have asunflower oil that has a balance of good saturates and good unsaturates,i.e. that is high in unsaturates but has sufficient saturates to be usedfor margarines or hardstock without high levels of hydrogenation, thusleading to no trans-fatty acids in the resulting product. Basically,there remains a need for a sunflower plant that can produce seedcontaining oil which is high in oleic acid and in stearic acid withreduced linoleic levels.

SUMMARY OF THE INVENTION

It is therefore the object of the present invention to provide avegetable oil with high stearic acid (as saturated fatty acid) and higholeic acid (as unsaturated fatty acid) contents that will reduce theabove described problems with fat. In this oil the stearic acid shouldpreferably be in positions 1 and 3 of TAG.

The present invention is based on the following considerations. The seedfatty acid biosynthesis occurs inside the plastid (FIG. 2). A series ofcycling reactions catalysed by the enzymatic complex FAS I produces thepalmitoyl-ACP that has 16 carbons. A second enzymatic complex called FASII elongates the palmitoyl-ACP to stearoyl-ACP (18 carbons), that isfurther modified by the stearate desaturase to produce oleoyl-ACP. Theseare the three main fatty acids synthesised by the plastid, being cleavedoff the ACP by the action of the enzyme thioesterase and then exportedout of the plastid. Later in the cytoplasm, the oleic acid may bedesaturated to linoleic and linolenic acids.

The TAG (storage oil) is produced in the cytoplasm using the pool offatty acids in the cytoplasm. This fatty acid pool consists of the fattyacids exported from the plastid and the linoleic acid made in thecytoplasm by desaturation. Thus, the fatty acid composition of TAG isdetermined by the fatty acids exported out of the plastid plus thelinoleic acid produced in the cytoplasm.

It was then contemplated that a new plant that is rich in stearic andoleic acids could be selected if a reduced stearate desaturase activity(leading to a decrease in the amount of oleoyl-ACP formed and thereforein an increase in the stearoyl-ACP) was combined with a goodthioesterase activity on stearoyl-ACP (which leads to the stearic acidbeing transported out of the plastid into the cytoplasm). This plantwill produce an accumulation of stearoyl-ACP inside the plastid, and thegood activity of the thioesterase over stearoyl-ACP should export itvery well out of the plastid, having there a high stearic acid contentavailable for TAG biosynthesis.

Out of the plastid, in the cytoplasm the high oleic character isnecessary to keep the linoleic acid content low. In high oleic lines,the conversion pathway does not work properly, so there is no conversionof oleic acid to linoleic acid.

The present invention is thus based on the finding that by selection ofone parent line that has a high stearic (HS) acid content on the onehand and a second parent line having a high oleic and high thioesterase(HOHT) activity over stearoyl-ACP on the other hand, crosses can be madethat result in seeds having a combination of the high stearic and higholeic properties (HSHO). In addition, it was surprisingly found that insaid oil a maximum of 10 wt % of the fatty acid groups in the sn-2position of the TAG molecules are saturated fatty acid groups.

Therefore, the present invention relates to plant seeds that contain anoil comprising an oleic acid content of more than 40 wt % and a stearicacid content of more than 12 wt % based on the total fatty acid contentof said oil, and wherein a maximum of 10 wt % of the fatty acid groupsin the sn-2 position of the TAG molecules constituting the oil aresaturated fatty acid groups. Preferably, the saturated fatty acid groupsare stearic acid groups. It is preferred that the oil has in the sn-2position of the TAG molecules a maximum of 8%, more preferably a maximumof 5 wt % of saturated fatty acid groups, in particular stearic acidgroups.

Regarding the other fatty acids, it is preferred that the oleic acidcontent is from 55 to 75 wt %, the stearic acid content is from 15 to 50wt %, in particular 20 to 40 wt %, and the linoleic acid content is lessthan 20 wt %. Preferably the total level of saturated fatty acids is atleast 20 wt %.

Selection of the parents can be achieved as follows.

Lines with high stearic acid content are lines having a stearic acidcontent of more than 12%, preferably more than 20%. One example of sucha high stearic (HS) parent line, which was selected after mutagenesisand has a stearic acid content of 26 wt %, is available as “CAS-3” (ATCCdeposit no. 75968, deposited on Dec. 14, 1994). Another example is“CAS-4”, having a stearic acid content of 16.1 wt % (ATCC deposit no.75969, deposited on Dec. 14, 1994) . By analysing the fatty acidcomposition of oil derived from the seeds of other candidate lines, theskilled person will be able to select other suitable parent lines.

It was found that some of the usual high oleic varieties could not beused for the purpose of the invention because they were found to havevery low thioesterase activity over the stearoyl-ACP. To overcome this,by measuring the thioesterase activity, lines with good activity overstearoyl-ACP can be selected from the available high oleic linescollections.

In short, one would first analyse the fatty acid composition of the oilof several promising lines. A suitable HOHT parent line would have morethan 7–8% stearic acid and either less than 5% linoleic acid or morethan 75% oleic acid. Subsequently, the selected lines must be grown andself pollinated. The total thioesterase activity is measured in seeds 15days after flowering (15DAF) on both oleoyl-ACP and stearoyl-ACP. Insuitable lines, the activity over stearoyl-ACP should be more than 10%of the activity over oleoyl-ACP. The ratio between both activitiesdetermines whether a line is suitable as a parent line or not.

In Table 2 the fatty acid composition and thioesterase activity of twohigh oleic sunflower lines are illustrated.

TABLE 2 Stearic acid content and thioesterase Vmax over the stearoyl-ACPof 15 days after flowering seeds from two high oleic sunflower lines.Stearic acid Thioesterase activity Sunflower line (%) Vmax HOHT 17.82.03 HOLT 8.0 0.82The HOHT line is a high oleic line with thioesterase over stearoyl-ACPactivity (HOHT) of more than twice the thioesterase Vmax overstearoyl-ACP than an usual high oleic line (HOLT). The relative activityof the enzymes over the stearoyl-ACP standardised with respect to theone over oleoyl-ACP is illustrated in FIG. 3. This line has as aconsequence more stearic acid at 15 days after flowering (Table 2) andalso in the oil obtained from the mature seed (Table 3).

TABLE 3 Fatty acid composition (%) of seeds from two high oleicsunflower lines. Fatty acid composition (%) Sunflower line palmiticstearic oleic linoleic araquic behenic HOHT 4.3 9.7 78.5 3.9 1.0 2.6HOLT 3.8 4.9 84.3 4.8 0.5 1.7This HOHT parent line was deposited on Sep. 7, 1999 with the AmericanType Culture Collection (10801 University Boulevard, Manassas, Va.20110-2209) and was assigned the number PTA-628.

Lines of both types (HOHT and HOLT) have been crossed with the highstearic CAS-3 line. In FIG. 4 (for HOHT) and 5 (for HOLT), the F2segregation for both traits (high stearic acid content and high oleicacid content) are shown. The seeds with higher content in stearic andoleic acids are within a circle. From the figures it follows that theHOHT line with high thioesterase activity over stearoyl-ACP has higholeic high stearic seeds and the line without high thioesterase activityhas no seeds of this type. Table 4 shows the fatty acid composition ofthese lines.

TABLE 4 Fatty acid composition of selected high oleic and stearic lines,with high and low thioesterase activity over stearoyl-ACP, aftercrossing with HS line CAS-3 Fatty acid composition (%) Sunflower linepalmitic stearic oleic linoleic araquic behenic HOHTxCAS-3 5.2 24.6 59.26.8 1.8 2.4 HOLTxCAS-3 4.3 17.4 72.1 4.0 1.3 2.8

The selected F2 lines are selfed for 5 to 6 generations in isolatedconditions to avoid contamination. The resultant generations areselected, based on high oleic and stearic acid content. Thioesteraseactivity can be analysed to assist in the selection process. Likewise,marker assisted breeding can be employed to track any or all of thethree traits to make the selection process quicker. Various markers suchas SSR microsatellite, ASO, RFLP and likewise can be employed. The useof markers is not necessary, as standard tests are known for determiningoleic, stearic, and thioesterase activity. However, once identifiedmarkers make trait tracking easier and earlier in the plant's life.

The true breeding plants produce an oil having a similar fatty acidcomposition to the F2 seeds selected with a low content of saturatedfatty acid in the 2 position of the TAG molecule (Table 5).

TABLE 5 Fatty acid composition of oil, TAG and sn-positions of truebreeding HSHO plants selected. n.d. = not detected. Fatty acidcomposition (mol %) Palmitic Stearic Oleic Linoleic Araquic BehenicTotal oil 5.5 24.9 57.8 8.2 1.7 1.8 TAG 5.6 26.1 57.6 7.4 1.6 1.7 sn-2position 1.7 1.9 87.4 9.0 n.d. n.d. sn-1 and 3 7.2 33.1 46.8 7.3 2.7 2.9position

The invention also relates to plants which form seeds which contain theabove described oil of the invention and to the oil per se as well as toproducts derived from the seeds, such as meal and crushed seeds. Theplants, seeds, oil, meal and crushed seeds of the invention are forexample sunflower plants, seeds, oil, meal and crushed seeds.

The plants and seeds of the invention are obtainable by a methodcomprising:

-   -   a) providing seeds which contain an oil having its a stearic        acid content of at least 3.2 wt % based on the total fatty acid        content of the oil;    -   b) providing seeds which contain an oil having an oleic acid        content of at least 40 wt % based on the total fatty acid        content of the oil, and which have a thioesterase activity over        stearoyl-ACP that is at least 10% of the thioesterase activity        over oleoyl-ACP;    -   c) crossing plants grown from the seeds provided in step (a) and        (b);    -   d) harvesting the F1 seed progeny.

Preferably, the method further comprises the steps of:

-   -   e) planting the F1 progeny seeds to grow plants;    -   f) self-pollinating the plants thus grown to produce F2 seed;    -   g) testing the seed for the presence of a stearic acid content        in the oil of at least 12 wt % and an oleic acid content of at        least 40 wt % and a thioesterase activity over stearoyl-ACP that        is at least 10% of the thioesterase activity over oleoyl-ACP;    -   h) planting seeds having the desired levels of stearic acid        content, oleic acid content and thioesterase activity to grow        plants;    -   i) self-pollinating the plants thus grown to produce F3 seed;        and    -   j) optionally repeating steps g), h) and i) until the desired        levels of stearic acid content, oleic acid content and        thioesterase activity are fixed.

Preferably, the stearic acid content is at least 15 wt %, preferably atleast 20 wt %.

The present invention also covers the method of obtaining an oil, inparticular a sunflower oil, having an oleic acid content of more than 40wt % and a stearic acid content of more than 12 wt % based on the totalfatty acid content of the oil by extracting oil from the seeds. Themethod preferably includes an extraction process which does not involvea substantial modification of the (sunflower) oil.

Additionally, in the process of extraction of the oil from the seedsthere is preferably no substantial chemical or physical modification norenzymatic rearrangement taking place and preferably no substantialhardening of the oil.

The present invention also includes food products comprising oilobtainable from seeds, in particular sunflower seeds, having an oleicacid content of more than 40 wt % and a stearic acid content of morethan 12 wt % based on the total fatty acid content of the oil. Foodproducts that are particularly useful for this type of oil includespreads, margarines, shortenings, sauces, ice-cream, soups, bakeryproducts, confectionery products, and the like. In these food productsthe level of (sunflower) oil is preferably from 3 to 100 wt % relativeto the total oil weight in the product. When used to form a spreadaccording to the present invention the (sunflower) oil is preferablyused as a hardstock at levels of 5 to 20 wt %.

The sunflower seeds of the present invention are also suitable per sefor human and animal consumption.

The present invention also encompasses cosmetic products comprising anoil, in particular a sunflower oil, the oil having an oleic acid contentof more than 40 wt % and a stearic acid content of more than 12 wt %based on the total fatty acid content of the oil. These cosmeticproducts can preferably contain levels of (sunflower) oil from 3 to 100wt %. Some examples of these cosmetic products would include creams,lotions, lipsticks, soap bars and skin or hair oils.

The present invention also includes a process for selecting Helianthusannuus plants, capable of producing seeds having the desired oil. Thesteps of the method are a) selecting a number of Helianthus annuusplants, collecting therefrom the seeds, the oil of which has a stearicacid content of at least 12 wt % and preferably 18 wt % based on thetotal fatty acid content; (b) selecting a number of Helianthus annuusplants, collecting therefrom the seeds, which express an oleic acidcontent of at least 40 wt % based on the oil present in the seed and athioesterase activity over stearoyl-ACP that is at least 10% of thethioesterase activity over oleoyl-ACP; (c) crossing the plants grownfrom the seeds of (a) and (b); and, harvesting the F1 seed progeny.

Additional steps include the steps of: (d) planting of the seeds orembryo rescue of the embryos of the F1 progeny obtained to form F2segregating seeds; (e) selecting from the F2 seeds which developedplants, those plants which produce seeds having an oleic acid content ofmore than 40 wt % and a stearic acid content of more than 12 wt % basedon the total fatty acid content of the oil, optionally selfing theselected plant to form true breeding inbreds.

The present invention also includes the process for producing F1 hybridseed. The steps of the method are a) planting seed of two inbreds havinghigh oleic acid content of at least 40 wt % and thioesterase activityover stearoyl-ACP that is at least 10% of the thioesterase activity overoleoyl-ACP, one of which may be male sterile, b) crossing the twoinbreds, and c) harvesting the F1 seed capable of producing F2 seed withan at least 40 wt % oleic acid content and an at least 12 wt % stearicacid content.

The present invention encompasses a vegetable oil with a new and uniquefatty acid composition produced in easy to grow crops. The preferredcrop is sunflower. This plant was used for making this invention.However, the invention is more broadly applicable and selection ofsuitable parents to produce the derived vegetable oil could likewisemodify other crops. These crops would include at least Brassicas,peanuts, palms and other oil producing plants. When mutation is used formaking one or both of the parents, the crop should be susceptible tomutagenically induced oil changes. Rape seed meets all theserequirements as does sunflower, these crops are presently some of themost useful crops for production of this new and unique fatty acidcomposition in the oil of their seeds.

BRIEF DESCRIPTION OF THE DRAWINGS

In this application reference is made to the following figures:

FIG. 1: hydrolysis of triacylgycerols by lipase;

FIG. 2: plastid showing the fatty acid biosynthesis in oilseeds;

FIG. 3: elevated thioesterase activity shown as the relative activity ofthe thioesterase over stearoyl-ACP and oleoyl-ACP of HOHT and HOLT;

FIG. 4: the F2 segregation for stearic and oleic acids of the crossbetween high oleic with high thioesterase activity over stearoyl-ACPline (HOHT) and a high stearic acid line (CAS-3);

FIG. 5: the F2 segregation for stearic and oleic acids of the crossbetween high oleic with low thioesterase activity over stearoyl-ACP line(HOLT) and a high stearic acid line (CAS-3).

DETAILED DESCRIPTION OF THE INVENTION

Definitions

“SUNFLOWER” shall mean Helianthus annuus.

“PLANT” shall include the complete plant and all plant and cell partsincluding pollen, kernel, oil, embryo, stalk, head, roots, cells,meristems, ovule, anthers, microspores, embryos, DNA, RNA, petals,seeds, and the like and protoplasts, callus or suspensions of any of theabove.

“15DAF” shall mean 15 days after flowering.

“TOTAL FATTY ACID CONTENT” of the sunflower oil refers to the sum ofC16:0, 18:0, 18:1, 18:2, 20:0, 22:0 and the traces of other like fattyacids as determined simultaneously in the oil from the seed.

“HOLT” shall mean having high to medium-high (40%–90%) oleic acid levelsin the oil when compared to normal, wildtype sunflower seed (oleic acidlevels of 17%–20%) wherein there are “LOW LEVELS OF THIOESTERASEACTIVITY”. A “HOLT LINE” is a line, in particular a sunflower line,having the HOLT trait.

“HOHT” shall mean having high to medium-high (40%–90%) oleic acid levelsin the oil when compared to normal, wildtype sunflower seed (oleic acidlevels of 17%–20%) wherein there are “HIGH LEVELS OF THIOESTERASEACTIVITY”. A “HOHT LINE” is a line, in particular a sunflower line, thathas the HOHT trait.

“HIGH LEVELS OF THIOESTERASE ACTIVITY” shall mean levels (at 15DAF) ofthioesterase activity over stearoyl-ACP which are at least 10% of thethioesterase activity over oleoyl-ACP. Consequently, “LOW LEVELS OFTHIOESTERASE ACTIVITY” shall mean levels which are below the “HIGHLEVELS OF THIOESTERASE ACTIVITY”.

“HS” shall mean having stearic acid levels in the oil of at least 12 wt% and preferably at least 15 wt % or more preferably at least 18 wt % oreven at least 20 wt % based on the total fatty acid content. “HIGHSTEARIC LINE” or “HS LINE” shall mean a line, in particular a sunflowerline, having the HS trait.

“HOHS” shall mean having levels of above 40% oleic acid and at least 12wt % stearic acid in the oil and preferably having levels of at least15% wt, more preferably at least 18 wt % or even at least 20 wt %stearic acid in the oil. A “HOHS LINE” shall mean a line having the HOHStrait.

EXAMPLES

Introduction

Preparation of HS Parent

In order to obtain the HS parent a method can be used for preparingsunflower seeds having an increased stearic acid and oleic acid contentas compared to wild type seeds. This method includes the step oftreating parent seeds with a mutagenic agent during a period of time andin a concentration sufficient to induce one or more mutations in thegenetic trait involved in stearic acid or oleic acid biosynthesis. Thisresults in an increased production of stearic acid and/or an increasedlevel of oleic acid. These mutagenic agents include agents such assodium azide or an alkylating agent, like ethyl methane sulfonate, ofcourse any other mutagenic agent having the same or similar effects mayalso be used. The treated seeds will contain inheritable geneticchanges. These mutated seeds are then germinated and progeny plants aredeveloped therefrom. To increase the traits in the lines the progeny canbe crossed or selfed. The progeny seeds are collected and analysed.

Sodium azide and ethyl methane sulfonate were used as mutagenic agentsin Example 1. Several sunflower lines with a stearic acid contentbetween 12 and 45% have been obtained. In all these cases the originalsunflower parent line for the production of the high stearic acid linesused was RDF-1–532 (Sunflower Collection of Instituto de AgriculturaSostenible, CSIC, Cordoba, Spain) that has from 4 to 7% stearic acidcontent in the seed oil.

Selecting the HOHT Parent

In principle it is sufficient to screen oleic lines for a HOHT phenotypeand use this line for either transformation or for crossing to a highstearic line to develop a HOHS line. A suitable line is at least theHOHT parent line that was deposited on Sep. 7, 1999 with the AmericanType Culture Collection (10801 University Boulevard, Manassas, Va.20110-2209) and was assigned the number PTA-628.

Making the HOHS Line

Seeds having the HOHT trait or the stearic trait can then be crossed toeach other to form the HOHS line. Optionally there can be additionalcycles of germination, culturing, and selfing to fix the homozygosity ofthe traits in the lines and crossing and collection of seeds.

Materials and Methods

Plants Growth Conditions

Sunflower (Helianthus annuus L.) seeds from high oleic lines withaltered seed fatty acid content was used to test for the thioesteraseactivities over stearoyl-ACP. Plants were cultivated in growth chambersat 25/150° C. (day/night) temperature, 16 hours photoperiod and photonflux density of 300 micromol m⁻²s⁻¹. Seeds for analysis were harvestedat 15 days after flowering and kept at −20° C.

Radioactive Reagents and Preparation of Acyl-ACPs

1-¹⁴C-Oleic with specific radioactivity of 2.1 GBq/mmol and (9,10(n)-³H) stearic acid with specific radioactivity of 1.9 GBq/mmol wereobtained from American Radiolabeled Chemicals Inc. (St. Louis, Mo.,USA). To prepare the fatty acid sodium salt, an appropriate volume offatty acid solution was transferred to a glass tube, the solvent wasremoved under a stream of nitrogen, and the residue was dissolved in 10%Triton X-100, 0.6 mM NaOH. This solution was heated at 55° C. for 1 hourto ensure homogeneity.

Acyl-ACPs were prepared using a modification of the enzymatic synthesisprocedure of Rock C.O. et al. (1981) Methods Enzymology 72:397–403.Assays contained 0.1 M Tris-HCl (pH 8.0), 0.4 M LiCl, 5 mM ATP, 10 mMMgCl₂, 2 mM DTT, 130 microM fatty acid sodium salt, 0.27 mM ACP-SH and1.8 mU of acyl-ACP synthetase (the last two components were purchasedfrom Sigma-Aldrich Quimica S.A. Madrid, Spain) in a final volume of 110microliter. Reactions were incubated at 37° C. for 3 hours. After thistime the pH was acidified to 6.0 by adding 1 microliter of 3.6 M HCl andthe mixture was cleaned of free fatty acids using a modification of themethod described by Mancha M. et al. ((1975) Anal. Biochem. 68:600–608),which method consists of adding an equal volume of isopropanol andwashing three times with hexane saturated in water/isopropanol (1:1;v/v).

Preparation of Crude Extracts for Enzyme Assays and ProteinDetermination

Frozen seeds were peeled and ground in extract buffer containing 20 mMTris-HCl (pH 8.5), 2 mM DTT and 5% (v/v) glycerol (Dörmann P. et al.(1994) Biochim. Biophys. Acta 1212:134–136) at 1 g of tissues/10 ml ofbuffer. Protein concentrations were measured using a Protein Assay Kit(Bio-Rad) according to the manufacturer's recommendations, with BSA asstandard.

Enzyme Assays

Acyl-ACP thioesterase activity was assayed in a final volume of 170microliter using 130 microliter of crude extract. Control assays hadcrude extract omitted. Reactions mixtures contained 20 mM Tris-HCl (pH8.5), 5% glycerol and 2 mM dithiothreitol (DTT) and differentconcentrations of substrates (stearoyl-ACP and oleoyl-ACP). Incubationswere carried out for 20 min at 25° C. Reactions were stopped by theaddition of 170 microliter of 1 M acetic acid in isopropanol containing1 mM of oleic acid. Mixtures were then washed three times with hexanesaturated in water/isopropanol (1:1, v/v).

Acyl-ACP thioesterase activity was determined by counting theradioactivity of the aqueous phase, which contained the non-hydrolysedsubstrates. Then, 3 ml of solvent scintillant (purchased from NationalDiagnostics, Hessle, England) was added and the radioactivity wasmeasured using a scintillation counter (Rackbeta II; LKB, Sweden). Datafrom acyl-ACP thioesterase assays were fitted to the Michaelis-Mentenequation by non-linear least-squares regression analysis using MicrocalOrigin 4.1, and correlated to P<0.05, as determined by paired Student'stest. Vmax and Km were derived from these curves.

Example 1

Preparation of a HS Line

1. Mutation with EMS

Seeds were mutagenised with a solution of 70 mM of ethyl methanesulfonate (EMS) in water. The treatment was performed at roomtemperature during 2 hours while shaking (60 rpm). After mutagenesis theEMS solution was discarded and seeds were washed during 16 hours undertap water.

Treated seeds were germinated in the field and plants wereself-pollinated. The seeds collected from these plants were used toselect new sunflower lines with modifications in the fatty acidcomposition. By using the method of Garcés, R. and Mancha, M. ((1993)Anal. Biochem. 211, 139–143) the seed fatty acid composition wasdetermined by gas liquid chromatography, after converting the fattyacids into their corresponding methyl esters.

A first plant with 9 to 17% stearic acid content in the oil wasselected. The progeny was cultivated for five generations wherein thestearic acid content increased and the new genetic trait became stablyfixed in the genetic material of the seed. This line is called CAS-3.The minimum and the maximum stearic acid content of the line were 19 and35% respectively. The stearic acid content of oil extracted from seedsfrom this cell line may thus lie between 19 and 35%.

2. Mutation with Sodium Azide

Sunflower seeds were mutagenised with sodium azide, at a concentrationof 2 mM in water. The treatment was performed at room temperature duringtwo hours while shaking (60 rpm). Then the mutagenesis solution wasdiscarded and seeds were washed during 16 hours with tap water.

Seeds were planted in the field and plants were self-pollinated. Seedsfrom these plants were collected, and the fatty acid composition wasdetermined by gas liquid chromatography, after converting the fattyacids into their corresponding methyl esters using the method describedin Example 1.

Seeds from a plant having around 10% stearic acid in the oil wereselected and cultivated for five generations. During this procedure thestearic acid content was increased and the new genetic trait fixed. Thisline is called CAS-4. A selected sample of this line was analysedresulting in a stearic acid content of 16.1%. The minimum and themaximum values were 12 and 19%, respectively.

TABLE 6 Percentage fatty acids Line Palmitic Stearic Oleic LinoleicCAS-3 5.1 26.0 13.8 55.1 CAS-4 5.5 16.1 24.3 54.1CAS-3 and CAS-4 are on deposit with the American Type CultureCollection, having ATCC numbers 75968 and 75969, respectively.

Example 2

Production of a HSHO Line

1. General

Sunflower plants were grown from the sunflower seeds of the HOHT line,seeds of which are on deposited at ATCC (PTA-628). Sunflower plants werealso grown from the sunflower seeds of CAS-3. The lines were crossed.The plants were assisted by artificial pollination in order to ensureadequate seed production occurred. The F1 seed was produced on the HOHTline, or vice versa, and harvested. The F2 seeds with more than 20%stearate and more than 40% oleate were selected. Although this producesthe oil of the present invention the level of production is limited.

Therefore fixed inbred lines evidencing seeds with these oil profilesare desirable. These homozygous fixed inbred HSHO lines can then becrossed to form hybrid seed, which will produce F2 seed evidencing thedesired oil traits of the present invention.

Toward this end the F1 seeds were planted and produced plants wereselfed in isolated conditions and F2 seed was produced. The F2 seed wastested for the three traits, high stearic, high oleic and high levels ofthioesterase activity. The remaining portion of the seeds evidencingthese traits was employed to grow plants to form F3 seed. The selfingand screening and selection process is repeated to develop the fixedhomozygous HSHO line, having the following fatty acid profile, C:16 5.4,C, 18.0 24.8, C, 18.1 58.5, C, 18.2 7.2. Once the trait is fixed similarHSHO lines can cross to form hybrid seed having both traits.

According to the invention sunflower plants and seeds from which saidoil can be extracted have been obtained by means of a biotechnologicalprocess. This high stearic acid content is an inheritable trait and isfairly independent from the growing conditions.

2. First Cross

A sunflower plant was grown from a sunflower seed of an HOHT line havinga stearic acid content of 10.7 wt % and an oleic acid content of 74.6 wt%. A sunflower plant was also grown from a CAS-3 sunflower seed. Theplants were crossed. The plants were assisted by artificiallypollination in order to ensure adequate seed production occurred. The F1seed was produced on the HOHT line, or vice versa, and harvested.

A F1 seed having a stearic acid content of 9.8 wt % and an oleic acidcontent of 80.7 wt %, was selected. This F1 seed was planted andproduced a plant which was selfed in isolated conditions and F2 seedswere produced. These F2 seeds were tested for oleic and stearic acidcontents. A seed containing 23.6 wt % of stearic acid and 65.5 wt % ofoleic acid was selected.

This F2 seed was planted and produced a plant which was selfed inisolated conditions and at 15DAF several seeds were collected andanalysed for stearoyl-ACP thioesterase activity. Plants with seedsrendering more than 10% stearoyl-ACP thioesterase referred to theoleoyl-ACP thioesterase activity of the same plant were selected.

Mature seeds from the plants selected in the previous step and havingstearic acid content higher than 20 wt % and oleic acid content higherthan 40 wt % were submitted to the selfing, screening and selectionprocess repeatedly to develop the fixed homozygous high stearic higholeic line having the following fatty acid profile in the oil:

-   -   palmitic 7.8 wt %;    -   stearic 24 wt %;    -   oleic 57.7 wt %;    -   linoleic 5.9 wt %;    -   araquic 1.9 wt %;    -   behenic 2.7 wt %.        Once the trait is fixed, similar high stearic high oleic lines        can cross to form hybrid seed having the above selected traits.

An analysis of the sn-2 position and sn-1,3 positions of the TAGmolecules of this oil indicates the following distribution of fattyacids (in wt %):

sn-2:

-   -   palmitic 3.3%;    -   stearic 3.4%;    -   oleic 88.8%;    -   linoleic 4.5%;    -   araquic 0%;    -   behenic 0%        sn-1,3:    -   palmitic 9%;    -   stearic 29.9%;    -   oleic 51.1%;    -   linoleic 4.7%;    -   araquic 2.3%;    -   behenic 3%        Thus, the total amount of saturated fatty acid groups in the        sn-2 position of the TAG molecules of this oil is 6.7 wt %.        3. Second Cross

A sunflower plant was grown from a sunflower seed of an HOHT line havinga stearic acid content of 8.4 wt % and an oleic acid content of 78.5 wt%. A sunflower plant was also grown from a CAS-3 sunflower seed. Theplants were crossed. The plants were assisted by artificiallypollination in order to ensure adequate seed production occurred. The F1seed was produced on the HOHT line, or vice versa, and harvested. A F1seed having a stearic acid content of 7.1 wt % and an oleic acid contentof 84.6 wt %, was selected. This F1 seed was planted and produced aplant which was selfed in isolated conditions and F2 seeds wereproduced. These F2 seeds were tested for oleic and stearic acidcontents. A seed containing 22.8 wt % of stearic acid and 64.8 wt % ofoleic acid was selected.

This F2 seed was planted and produced a plant which was selfed inisolated conditions and at 15 DAF several seeds were collected andanalysed for stearoyl-ACP thioesterase activity. Plants with seedsrendering more than 10% stearoyl-ACP thioesterase referred to theoleoyl-ACP thioesterase activity of the same plant were selected. Matureseeds from the plants selected in the previous step and having stearicacid content higher than 20 wt % and oleic acid content higher than 40wt % were submitted to the selfing, screening and selection processrepeatedly to develop the fixed homozygous high stearic high oleic linehaving the following fatty acid profile in the oil:

-   -   palmitic 5.8 wt %;    -   stearic 24, 7 wt %;    -   oleic 57.6 wt %;    -   linoleic 8.2 wt %;    -   araquic 1.8 wt %;    -   behenic 1.9 wt %.        Once the trait is fixed, similar high stearic high oleic lines        can cross to form hybrid seed having the above selected traits.

An analysis of the sn-2 position and sn-1,3 positions of the TAGmolecules of this oil indicates the following distribution of fattyacids (in wt %):

sn-2:

-   -   palmitic 1.7%;    -   stearic 1.9%;    -   oleic 87.5%;    -   linoleic 8.9%;    -   araquic 0%;    -   behenic 0%        sn-1,3:    -   palmitic 7.2%;    -   stearic 33.2%;    -   oleic 46.9%;    -   linoleic 7.3%;    -   araquic 2.6%;    -   behenic 2.8%.        Thus, the total amount of saturated fatty acid groups in the        sn-2 position of the TAG molecules of this oil is 3.6 wt %.        4. Third Cross

A sunflower plant was grown from a sunflower seed of an HOHT line havinga stearic acid content of 9.9 wt % and an oleic acid content of 81.2 wt%. A sunflower plant was also grown from a CAS-3 sunflower seed. Theplants were crossed. The plants were assisted by artificiallypollination in order to ensure adequate seed production occurred. The F1seed was produced on the HOHT line, or vice versa, and harvested.

A F1 seed having a stearic acid content of 8.9 wt % and an oleic acidcontent of 82.3 wt %, was selected. This F1 seed was planted andproduced a plant which was selfed in isolated conditions and F2 seedswere produced. These F2 seeds were tested for oleic and stearic acidcontents. A seed containing 23.9 wt % of stearic acid and 64.0 wt % ofoleic acid was selected.

This F2 seed was planted and produced a plant which was selfed inisolated conditions and at 15 DAF several seeds were collected andanalysed for stearoyl-ACP thioesterase activity. Plants with seedsrendering more than 10% stearoyl-ACP thioesterase referred to theoleoyl-ACP thioesterase activity of the same plant were selected. Matureseeds from the plants selected in the previous step and having stearicacid content higher than 20 wt % and oleic acid content higher than 40wt % were submitted to the selfing, screening and selection processrepeatedly to develop the fixed homozygous high stearic high oleic linehaving the following fatty acid profile in the oil:

-   -   palmitic 5.4 wt %;    -   stearic 24, 2 wt %;    -   oleic 62.1 wt %;    -   linoleic 4.7 wt %;    -   araquic 1.6 wt %;    -   behenic 2.0 wt %.        Once the trait is fixed, similar high stearic high oleic lines        can cross to form hybrid seed having the above selected traits.

An analysis of the sn-2 position and sn-1,3 positions of the TAGmolecules of this oil indicates the following distribution of fattyacids (in wt %):

sn-2:

-   -   palmitic 1.8%;    -   stearic 3.3%;    -   oleic 89.6%;    -   linoleic 5.3%;    -   araquic 0%;    -   behenic 0%        sn-1,3:    -   palmitic 9.5%;    -   stearic 33.5%;    -   oleic 48.2%;    -   linoleic 4.3%;    -   araquic 2.2%;    -   behenic 2.3%        Thus, the total amount of saturated fatty acid groups in the        sn-2 position of the TAG molecules of this oil is 5.1 wt %.

The present application pertains to genetic material, comprising plantseeds, which include the oil contained therein, meal and crushed seeds,as well as the process of growing the seeds and the plants that are theresult from growing the seeds and plants producing the seeds.

1. Sunflower seeds that contain oil having an oleic acid content of morethan 40 wt % and a stearic acid content of more than 12 wt % based onthe total fatty acid content of said oil, wherein a maximum of 10 wt %of the fatty acid groups in the sn-2 position of the triacylglycerolmolecules are saturated fatty acids, and wherein the oil has a linoleicacid content of less than 20 wt %.
 2. Sunflower seeds according to claim1, wherein the seeds contain an oil that has in the sn-2 position of thetriacylglycerol molecules constituting the oil a maximum of 8 wt % ofsaturated fatty acid groups.
 3. Sunflower seeds according to claim 2,wherein the seeds contain an oil that has in the sn-2 position of thetriacylglycerol molecules constituting the oil a maximum of 5 wt % ofsaturated fatty acid groups.
 4. Sunflower seeds according to claim 1,wherein the oleic acid content is from 55 to 75 wt %.
 5. Sunflower seedsaccording to claim 1, wherein the stearic acid content is from 15 to 50wt %.
 6. Sunflower seeds according to claim 5, wherein the stearic acidcontent is from 20 to 40 wt %.
 7. Sunflower seeds according to claim 1,wherein the oil has a total level of saturated fatty acids of at least20 wt %.
 8. Method for producing a sunflower plant which forms seeds asclaimed in claim 1, which method comprises: a) providing sunflower seedswhich contain oil having a stearic acid content of at least 12 wt %; b)providing sunflower seeds which contain an oil having an oleic acidcontent of at least 40 wt % and a thioesterase activity overstearoyl-ACP of at least 10% of the thioesterase activity overoleoyl-ACP; c) crossing sunflower plants grown from the sunflower seedsprovided in step a) and b); d) harvesting the F1 seed progeny.
 9. Methodas claimed in claim 8, further comprising the steps of: e) planting theF1 progeny seeds to grow sunflower plants; f) self-pollinating thesunflower plants thus grown to produce F2 seed; g) testing the seed forthe presence of a stearic acid content of at least 12 wt %, an oleicacid content of at least 40 wt % and a thioesterase activity overstearoyl-ACP of at least 10% of the thioesterase activity overoleoyl-ACP; h) planting sunflower seeds having a stearic acid content ofat least 12 wt %, an oleic acid content of at least 40 wt %, and athioesterase activity over stearoyl-ACP of at least 10% of thethioesterase activity over oleoyl-ACP to grow sunflower plants; i)self-pollinating the sunflower plants thus grown to produce F3 seed; andj) optionally repeating steps g), h) and i) until the stearic acidcontent of at least 12 wt %, the oleic acid content of at least 40 wt %,and the thioesterase activity over stearoyl-ACP of at least 10% of thethioesterase activity over oleoyl-ACP are fixed.
 10. Method as claimedin claim 8, wherein the seeds which contain oil having a stearic acidcontent of at least 12 wt % are provided by: a) mutagenic treatment ofsunflower seeds having a stearic acid content of less than 12%; b)producing sunflower plants therefrom which are pollinated to produceseeds; c) testing the seeds for the desired stearic acid content; and d)optionally repeating steps b) and c).
 11. Sunflower plants obtained bythe method of claim
 8. 12. Sunflower plants obtained by the method ofclaim
 9. 13. Sunflower plants obtained by the method of claim 10.